Inhibition of the CmeABC efflux pump by antisense peptide nucleic acids reduces the emergence of spontaneous fluoroquinolone resistant mutants in Campylobacter jejuni

Abstract

<p>Campylobacter is a major cause of bacterial gastroenteritis in humans and is commonly transmitted via undercooked poultry meat, unpasteurized milk and contaminated water. Over the years, Campylobacter has developed resistance to clinically important antibiotics including fluoroquinolones. Because of its significance in public health, both the Centers for Disease Control and Prevention and the World Health Organization has listed antibiotic resistant Campylobacter as a serious threat. Fluoroquinolone resistant mutants in Campylobacter occurs spontaneously and the resistance is mediated by the synergistic effect of the CmeABC multi-drug efflux pump and point mutations in the gyrA gene. Thus, antisense inhibition of cmeABC expression is a promising approach to combat fluoroquinolone resistance in Campylobacter. Previous studies have shown the specific inhibition of cmeABC expression by an antisense peptide nucleic acid (PNA) targeting the translational start of CmeA, but whether the PNA can be used as an adjuvant to potentiate fluoroquinolone antibiotics remains to be determined. Toward this end, in vitro and in vivo experiments were conducted to examine the efficacy of the PNA in reducing the emergence of spontaneous fluoroquinolone resistant mutants during treatment with a fluoroquinolone antibiotic. When fluoroquinolone-susceptible Campylobacter was treated with ciprofloxacin in culture media, resistant mutants emerged quickly and eventually replaced the susceptible population. However, addition of the PNA to the ciprofloxacin treatment prevented the emergence of resistant mutants completely, indicating the potentiating effect of the PNA on ciprofloxacin. Based on the in vitro results, the efficacy of the PNA was further evaluated in vivo using a Campylobacter infected chicken model. When given by oral gavage, the PNA was able to reduce, but unable to completely eliminate the emergence of fluoroquinolone resistant mutants in Campylobacter in the intestinal tract of chickens treated with enrofloxacin. These results demonstrate the potentiating effect of the PNA on fluoroquinolone antibiotics and warrant additional studies to further optimize the anti-CmeABC approach as an adjunct therapy for antibiotic treatment of campylobacteriosis.</p>